Elevated CO2 Induces Root Defensive Mechanisms in Tomato Plants When Dealing with Ammonium Toxicity.

An adequate carbon supply is fundamental for plants to thrive under ammonium stress. In this work, we studied the mechanisms involved in tomato (Solanum lycopersicum L.) response to ammonium toxicity when grown under ambient or elevated CO2 conditions (400 or 800 p.p.m. CO2). Tomato roots were observed to be the primary organ dealing with ammonium nutrition. We therefore analyzed nitrogen (N) and carbon (C) metabolism in the roots, integrating the physiological response with transcriptomic regulation. Elevated levels of CO2 preferentially stimulated root growth despite the high ammonium content. The induction of anaplerotic enzymes from the tricarboxylic acid (TCA) cycle led to enhanced amino acid synthesis under ammonium nutrition. Furthermore, the root transcriptional response to ammonium toxicity was improved by CO2-enriched conditions, leading to higher expression of stress-related genes, as well as enhanced modulation of genes related to signaling, transcription, transport and hormone metabolism. Tomato roots exposed to ammonium stress also showed a defense-like transcriptional response according to the modulation of genes related to detoxification and secondary metabolism, involving principally terpenoid and phenolic compounds. These results indicate that increasing C supply allowed the co-ordinated regulation of root defense mechanisms when dealing with ammonium toxicity.

Impact of dimethylpyrazole-based nitrification inhibitors on soil-borne bacteria.

Nitrogen (N) input from fertilizers modifies the properties of agricultural soils as well as bacterial community diversity, composition and relationships. This can lead to negative impacts such as the deterioration of system multifunctionality, whose maintenance is critical to normal nutrient cycling. Synthetic nitrification inhibitors (NIs) can be combined with fertilizers to improve the efficiency of N use by reducing N losses. However, analysis of their effects on non-target bacteria are scarce. This study aimed to analyze the effect of applying the NIs DMPP and DMPSA on the whole bacterial community. Through 16S rRNA amplicon sequencing we determined the differences between samples in terms of microbial diversity, composition and co-occurrence networks. The application of DMPP and DMPSA exerted little impact on the abundance of the dominant phyla. Nevertheless, several significant shifts were detected in bacterial diversity, co-occurrence networks, and the abundance of particular taxa, where soil water content played a key role. For instance, the application of NIs intensified the negative impact of N fertilization on bacterial diversity under high water-filled pore spaces (WFPS) (>64%), reducing community diversity, whereas alpha-diversity was not affected at low WFPS (<55%). Interestingly, despite NIs are known to inhibit ammonia monooxygenase (AMO) enzyme, both NIs almost exclusively inhibited Nitrosomonas genera among AMO holding nitrifiers. Thus, Nitrosomonas showed abundance reductions of up to 47% (DMPP) and 66% (DMPSA). Nonetheless, non-target bacterial abundances also shifted with NI application. Notably, DMPSA application partially alleviated the negative effect of fertilization on soil multifunctionality. A remarkable increase in populations related to system multifunctionality, such as Armatimonadetes (up to +21%), Cyanobacteria (up to +30%) and Fibrobacteres (up to +25%) was observed when DMPSA was applied. NI application substantially influenced microbial associations by decreasing the complexity of co-occurrence networks, decreasing the total edges and node connectivity, and increasing path distances.

Response of Wheat Storage Proteins and Breadmaking Quality to Dimethylpyrazole-Based Nitrification Inhibitors under Different Nitrogen Fertilization Splitting Strategies.

Improving fertilizer nitrogen (N) use efficiency is essential to increase crop productivity and avoid environmental damage. This study was conducted during four crop cycles of winter wheat under humid Mediterranean conditions (Araba, northern Spain). The effects of N-fertilization splitting and the application of the nitrification inhibitors (NIs) 3,4-dimethylpyrazole phosphate (DMPP) and 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) as strategies to improve grain quality were examined. The hypothesis of this study was to test if the partial ammonium nutrition and the reduction of fertilizer losses presumably induced by the application of NIs can modify the grain gliadin and glutenin protein contents and the breadmaking quality (dough rheological properties). Among both NIs assayed, only DMPP showed a slight effect of decreasing the omega gliadin fraction, following splitting either two or three times, although this effect was dependent on the year and was not reflected in terms of dough extensibility. The slight decreases observed in grain quality in terms of dough strength and glutenin content induced by DMPP suggest that DMPSA is more promising in terms of maintaining grain quality. Nonetheless, these poor effects exerted by NI application on grain quality parameters did not lead to changes in the quality parameters defining the flour aptitudes for breadmaking.

Relationship between tillage management and DMPSA nitrification inhibitor efficiency.

Agricultural sustainability is compromised by nitrogen (N) losses caused by soil microbial activity. Nitrous oxide (N2O) is a potent greenhouse gas (GHG) produced as consequence of nitrification and denitrification processes in soils. Nitrification inhibitors (NI) as 3,4-dimethylpyrazole-succinic acid (DMPSA) are useful tools to reduce these N losses from fertilization. The objective of this work was to test the efficiency of DMPSA in two different tillage management systems, conventional tillage (CT) and no-tillage (NT), in a winter wheat crop under Humid Mediterranean conditions. N fertilizer was applied as ammonium sulphate (AS) with or without DMPSA in a single or split application, including an unfertilized treatment. GHG fluxes (N2O, CO2 and CH4) were measured by the closed chamber method. amoA and nosZI genes were quantified by qPCR as indicators of nitrifying and denitrifying populations. Nitrification was inhibited by DMPSA in both CT and NT, while the higher water filled pore space (WFPS) in NT promoted a better efficiency of DMPSA in this system. This higher efficiency might be due to a greater N2O reduction to N2 as result of the nosZI gene induction. Consequently, DMPSA was able to reduce N2O emissions down to the unfertilized levels in NT. Provided that NT reduced CO2 emissions and maintained crop yield compared to CT, the application DMPSA under NT management is a promising strategy to increase agro-systems sustainability under Humid Mediterranean conditions.

A Multi-Species Analysis Defines Anaplerotic Enzymes and Amides as Metabolic Markers for Ammonium Nutrition.

Nitrate and ammonium are the main nitrogen sources in agricultural soils. In the last decade, ammonium (NH4 +), a double-sided metabolite, has attracted considerable attention by researchers. Its ubiquitous presence in plant metabolism and its metabolic energy economy for being assimilated contrast with its toxicity when present in high amounts in the external medium. Plant species can adopt different strategies to maintain NH4 + homeostasis, as the maximization of its compartmentalization and assimilation in organic compounds, primarily as amino acids and proteins. In the present study, we report an integrative metabolic response to ammonium nutrition of seven plant species, belonging to four different families: Gramineae (ryegrass, wheat, Brachypodium distachyon), Leguminosae (clover), Solanaceae (tomato), and Brassicaceae (oilseed rape, Arabidopsis thaliana). We use principal component analysis (PCA) and correlations among metabolic and biochemical data from 40 experimental conditions to understand the whole-plant response. The nature of main amino acids is analyzed among species, under the hypothesis that those Asn-accumulating species will show a better response to ammonium nutrition. Given the provision of carbon (C) skeletons is crucial for promotion of the nitrogen assimilation, the role of different anaplerotic enzymes is discussed in relation to ammonium nutrition at a whole-plant level. Among these enzymes, isocitrate dehydrogenase (ICDH) shows to be a good candidate to increase nitrogen assimilation in plants. Overall, metabolic adaptation of different carbon anaplerotic activities is linked with the preference to synthesize Asn or Gln in their organs. Lastly, glutamate dehydrogenase (GDH) reveals as an important enzyme to surpass C limitation during ammonium assimilation in roots, with a disparate collaboration of glutamine synthetase (GS).

Clover and ryegrass are tolerant species to ammonium nutrition.

The application of nitrification inhibitors (NIs) together with nitrogen fertilizers in grasslands is an effective alternative to reduce nitrate leaching and nitrogenous gases emissions to the atmosphere. Nevertheless, the use of NIs increases the amount of ammonium available for the plant that, due to its reported toxic effect in plants, can have a direct effect on crop production. Grassland species have traditionally suffered from intensive grazing and urea deposition and, therefore, a tolerance to ammonium nutrition could be expected in these species. Plants of Trifolium repens L. var. huia and Lolium perenne L. var. Herbus were grown under two nitrogen nutrition regimes (nitrate or ammonium) and three different nitrogen concentrations (0.5, 2.5 and 5 mmol/L). The effect of nitrogen form was determined on biomass production parameters, gas-exchange and water relations parameters as well as polyamine (PA) and ion tissue contents. Both grassland species showed tolerance to ammonium nutrition due to their capacity to adjust several metabolic processes in a species-specific way. Gas exchange measurements and biomass production (expressed as dry weight (DW)) were unaffected by the nitrogen form or dose in both species except for a decrease in root total DW in ryegrass plants grown under ammonium nutrition. Hydraulic conductance (L(0)) increased in ryegrass with increasing ammonium doses but no change due to the nitrogen source was observed in water potential (Psi(w)) values. Both species, and specially ryegrass, accumulated free ammonium mainly in roots when grown under ammonium nutrition and its translocation to the shoot via xylem was also observed. A clear difference in cations and PAs pattern was observed in each species when comparing both nitrogen nutrition regimes.

CO2 enrichment modulates ammonium nutrition in tomato adjusting carbon and nitrogen metabolism to stomatal conductance.

Ammonium (NH4(+)) toxicity typically occurs in plants exposed to high environmental NH4(+) concentration. NH4(+) assimilating capacity may act as a biochemical mechanism avoiding its toxic accumulation but requires a fine tuning between nitrogen assimilating enzymes and carbon anaplerotic routes. In this work, we hypothesized that extra C supply, exposing tomato plants cv. Agora Hybrid F1 to elevated atmospheric CO2, could improve photosynthetic process and thus ameliorate NH4(+) assimilation and tolerance. Plants were grown under nitrate (NO3(-)) or NH4(+) as N source (5-15mM), under two atmospheric CO2 levels, 400 and 800ppm. Growth and gas exchange parameters, (15)N isotopic signature, C and N metabolites and enzymatic activities were determined. Plants under 7.5mM N equally grew independently of the N source, while higher ammonium supply resulted toxic for growth. However, specific stomatal closure occurred in 7.5mM NH4(+)-fed plants under elevated CO2 improving water use efficiency (WUE) but compromising plant N status. Elevated CO2 annulled the induction of TCA anaplerotic enzymes observed at non-toxic NH4(+) nutrition under ambient CO2. Finally, CO2 enrichment benefited tomato growth under both nutritions, and although it did not alleviate tomato NH4(+) tolerance it did differentially regulate plant metabolism in N-source and -dose dependent manner.

Dicyandiamide and 3,4-dimethyl pyrazole phosphate decrease N2O emissions from grassland but dicyandiamide produces deleterious effects in clover.

The application of nitrogen fertilisers leads to different ecological problems such as nitrate leaching and the release of nitrogenous gases. N2O is a gas involved in global warming, therefore, agricultural soils can be regarded as a source of global warming. Soil N2O production comes from both the nitrification and denitrification processes. From an ecological viewpoint, using nitrification inhibitors with ammonium based fertilisers may be a potential management strategy to lower the fluxes of N2O, thus decreasing its undesirable effect. In this study, the nitrification inhibitors (NIs) dicyandiamide (DCD) and 3,4-dimethyl pyrazole phosphate (DMPP) have been evaluated as management tools to mitigate N2O emissions from mineral fertilisation and slurry application in grassland systems (experiments 1 and 2), and to assess the phytotoxic effect of these inhibitors per se on clover (experiment 3). Both nitrification inhibitors acted in maintaining soil nitrogen (N) in ammonium form, decreasing cumulative N2O emissions. DCD, but not DMPP, produced phytotoxic effects and yield reduction in white clover. A nutrient imbalance, which led to a senescence process visually observed as chlorosis and necrosis at the border of the leaves, was noted.

Root phosphoenolpyruvate carboxylase and NAD-malic enzymes activity increase the ammonium-assimilating capacity in tomato.

Plant ammonium tolerance has been associated with the capacity to accumulate large amounts of ammonium in the root vacuoles, to maintain carbohydrate synthesis and especially with the capacity of maintaining high levels of inorganic nitrogen assimilation in the roots. The tricarboxylic acid cycle (TCA) is considered a cornerstone in nitrogen metabolism, since it provides carbon skeletons for nitrogen assimilation. The hypothesis of this work was that the induction of anaplerotic routes of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and malic enzyme (NAD-ME) would enhance tolerance to ammonium nutrition. An experiment was established with tomato plants (Agora Hybrid F1) grown under different ammonium concentrations. Growth parameters, metabolite contents and enzymatic activities related to nitrogen and carbon metabolism were determined. Unlike other tomato cultivars, tomato Agora Hybrid F1 proved to be tolerant to ammonium nutrition. Ammonium was assimilated as a biochemical detoxification mechanism, thus leading to the accumulation of Gln and Asn as free amino acids in both leaves and roots as an innocuous and transitory store of nitrogen, in addition to protein synthesis. When the concentration of ammonium in the nutrient solution was high, the cyclic operation of the TCA cycle seemed to be interrupted and would operate in two interconnected branches to provide α-ketoglutarate for ammonium assimilation: one branch supported by malate accumulation and by the induction of anaplerotic PEPC and NAD-ME in roots and MDH in leaves, and the other branch supported by stored citrate in the precedent dark period.

High irradiance improves ammonium tolerance in wheat plants by increasing N assimilation.

Ammonium is a paradoxical nutrient ion. Despite being a common intermediate in plant metabolism whose oxidation state eliminates the need for its reduction in the plant cell, as occurs with nitrate, it can also result in toxicity symptoms. Several authors have reported that carbon enrichment in the root zone enhances the synthesis of carbon skeletons and, accordingly, increases the capacity for ammonium assimilation. In this work, we examined the hypothesis that increasing the photosynthetic photon flux density is a way to increase plant ammonium tolerance. Wheat plants were grown in a hydroponic system with two different N sources (10mM nitrate or 10mM ammonium) and with two different light intensity conditions (300 µmol photon m(-2)s(-1) and 700 µmol photon m(-2)s(-1)). The results show that, with respect to biomass yield, photosynthetic rate, shoot:root ratio and the root N isotopic signature, wheat behaves as a sensitive species to ammonium nutrition at the low light intensity, while at the high intensity, its tolerance is improved. This improvement is a consequence of a higher ammonium assimilation rate, as reflected by the higher amounts of amino acids and protein accumulated mainly in the roots, which was supported by higher tricarboxylic acid cycle activity. Glutamate dehydrogenase was a key root enzyme involved in the tolerance to ammonium, while glutamine synthetase activity was low and might not be enough for its assimilation.